SUMMARY Project 1 will use a translational research pipeline for human gut microbiota-directed diagnostics and therapeutics we developed based on gnotobiotic mice harboring microbiota transplanted from three types of adult twin pairs discordant for obesity and/or its associated metabolic abnormalities [LeanMetabolicallyHealthy-Obese Metabolically Unhealthy (LnMH/ObMUN), LnMH/ObMH, ObMH/ObMUN]. Its goal is to (i) establish a causal role of the gut microbiota in obesity and associated metabolic phenotypes, (ii) obtain mechanistic insights about the interactions between diet and members of the gut microbiota that produce these phenotypes, (iii) conduct tests of the effects of manipulating diet and bacterial taxa on phenotypes transmitted by ObMUN microbiota. Project 1 has 4 aims. (1) Determine the effects of microbiota, collected from twin pairs at the end of the each of their two in-home diet periods, on body composition/metabolic phenotypes of recipient adult gnotobiotic mice given a diet homologous to that consumed at the time the donor's microbiota was collected or the other diet that twins will have consumed (cross-over diet group). Within-pair, between-twin pair comparisons and across discordant pair type comparisons will be performed. (2) Co-house gnotobiotic mice harboring intact uncultured microbiota from discordant pairs to determine if (i) microbiota from the LnMH donor prevents or ameliorates development of obesity- and obesity-associated metabolic dysfunction in mice colonized with the ObMH or ObMUN co-twin's microbiota, and how prevention/amelioration correlates with invasion of bacterial taxa from the microbiota of one cagemate to the other, and later comparable co-housing experiments involving ObMH and ObMUN mice. (3) Determine if bacterial culture collections prepared from microbiota samples characterized in Aim 2 also transfer discordant donor phenotypes to gnotobiotic mice; perform co-housing experiments to identify invasive cultured taxa associated with phenotypic rescue in different diet contexts. (4) Execute a testing matrix in which a culture collection from a representative ObMUN or ObMH co-twin are introduced to separate groups of gnotobiotic mice fed a representative USA diet high in saturated fats and low in fruits and vegetables, alone or with a lead probiotic consortium (invasive taxa identified from aims 2, 3), or a lead prebiotic (identified from in vitro screen), or a combination of the two (synbiotic lead). The therapeutic lead will be administered at the time of colonization with the culture collection (prevention arm) or 2 weeks after initial colonization (treatment arm). Metabolic profiling will be performed with Core A. Multi-omics datasets will be analyzed with existing tools and used to generate new analysis strategies with Project 3 and Core B.